Interferons (IFN) are a family of proteins whose first described biological activity was the ability to inhibit viral replication. Besides their antiviral action, IFNs possess a variety of other biological activities, controlling cell growth, differentiation, and modulating the immune system (Vilcek, J. & Sem, G. C. In Virology (Ed.) Fields, B. N., Knipe, D. M., Howley, P. M. 375-399, PA: Lippincott-Raven Publishers, 1996).
Human IFNs are classified in two major sub-families based on their biological and physical properties. The type I IFN family includes fibroblast .beta.-interferon (IFN-.beta.), leukocyte a-interferon (IFN-.alpha.) with 15 or more genes and pseudo-genes and IFN-.omega. and IFN.tau.. The type 11 IFN family is represented by IFN-.gamma., produced by T lymphocytes and NK cells in response to mitogens and antigenic stimuli (Vilcek, J. & Sem, G. C. In Virology (Ed.) Fields, B. N., Knipe, D. M., Howley, P. M. 375-399, PA: Lippincott-Raven Publishers, 1996).
Like in humans, most of the mammalian species have large IFN-.alpha. gene families. The existence of multiple IFN-.beta. gene families was shown in bovine (three genes) whereas, humans and most of other mammalian species probably contain only one gene (Ryan, A. M., Gallagher, D. M., Womack, J. E Mammalian Genome, 3(19): 575-578,1992).
Placental tissues can produce IFNs. IFN activity has been detected in amniotic fluid, umbilical cord and blood of human placentae during pregnancy without clinical signs of viral infections (Lebon, P., Girard, S., Thepot, F. & Chany, C. Journal of General Virology, 59: 393-396, 1982) and also in mouse placental tissues (Fowler, A. K., Reed, C. D. & Giron, D. J. Nature, 285: 266-267, 1980). Ovine and bovine trophoblast cells produce another type of IFN named IFN-.tau., during the embryo implantation into the uterus (Imakawa, K., Anthony, R. V., Kazemi, M., Mariotti, H., Poltes, H. G. & Roberts, R. M. Nature, 330: 377-379, 1987.). This IFN is necessary for fetal implantation, acting as an anti-luteolysin, resulting in the continued secretion of progesterone (Cross, J. C. & Roberts, R. M. Proceedings of the National Academy of Sciences of the USA, 88: 3817-3820, 1991). Recently a human trophoblast IFN (hTIFN) cDNA was described by (Whaley, A. E., Meka, C. S. R., Harbison, L. A., Hunt, J. S. & Imakawa, K The Journal of Biological Chemistry, 269:10864-10868, 1994) whose nucleotide sequence is similar (85%) to ovine and bovine IFN-.tau.. The HulFN.tau. and HulFN-.omega. mRNAs were detected in the cytotrophoblast cells throughout the pregnancy. This fact suggests that these IFNs are important as a first line of defence, protecting the fetus from virus transmission and also participating in the progressive changes of placental development (Whaley, A. E., Meka, C. S. R., Harbison, L. A., Hunt, J. S. & Imakawa, K The Journal of Biological Chemistry, 269:10864-10868, 1994). Human trophoblast cultures produced IFN-.beta., IFN-.alpha.I and tau interferon when infected by virus (Aboagye-Mathiesen, G., Thoth, F. D., Juhl, C. H., Norskov-Lauritsen, N. N., Petersen, P. M. & Ebbesen, P. Journal of General Virology, 71: 3061-3066, 1990 and Thoth, F. D., Norskov-Lauritsen, N. N., Juhl, C. H, Aboagye-Mathiesen, G. & Ebbesen, P. Journal of General Virology, 71: 3067-3069, 1990).
IFNs bind to receptors located at the cellular surface, activating in this way the transduction of cytoplasmic signals that will induce in the nucleus genes responsible for the activities of IFN (JOKLIK, W. K., Interferons, In: FIELDS, B. N. AND KNIPE, D. M. (Ed.) Virology, 2a. Ed.--New York, Raven Press Ltd., cap.16, p.383-410,1990).
All the members of the family of IFNs .alpha. possess a sequence of 165 to 166 a.a., of the which 4 cysteines (Cys) are highly conserved in the positions 1; 29; 98; 99 or 100; 138 or 139; and they are responsible for disulfide bonds (S--S). The S--S bonds between Cys 1; 98; 99 or 100 and between Cys 29 and 138 or 139 are important in the stabilization of the IFN structure (WETZEL, R. et al., J. Interferon Res., 1:381-389,1981). The bond among these two Cys is indispensable in the maintenance of the biological activity of this IFN (PATH, T., EMBO J., 11: 3193-3201 et al., 1992).
IFN .beta. is produced by fibroblasts and it is a glycoprotein with relative molecular mass of 22-23 kDa and specific antiviral activity of 2-5.times.10.sup.8 international units (protein Ul)/mg. A gene located at chromosome 9 codes this 166 a.a IFN that doesn't possess introns. IFN .beta. of bovine, swine and caprine species are coded by multiple genes (OF MAEYER, E. M. and OF MAEYER-GUIGNARD, J.--Interferon and other regulatory cytokines, John Wiley and Sounds, New York, 1988).
IFN .beta. possesses 3 cysteine residues (Cys) in the positions 17, 31 and 141. Two Cys 31 and 141 form a disulfide bond while Cys 17 stays free. The studies (MARK, D. F., et al., Proc. Natl. Acad. Sci. It USES, 81: 5662-5666, 1984), in which the Cys 17 was changed for a serine showed that this IFN presented the same spectrum of biological activities of IFN .beta., such as anticellular and antiproliferative activity, activation of NK cells and neutralization of anti HuIFN antibodies, and presents a greater stability than natural HulFN .beta. when incubated at -70.degree. C.
In other experiments, SHEPARD, H. M. et al., Nature, 294:563-565, 1981, changed the Cys 141 for a tyrosine, rendering a biologically inactive molecule.
The mechanisms through which IFNs induce the antiviral state have been delineated. It is accepted that the activation of the enzyme 2'5' oligoadenilate synthethase and of the RNA dependent protein kinase (dsRNA) are important in the antiviral action (PESTKA, S., et al., Ann. Rev. Biochem., 56:727-777, 1987).
It is an object of the present invention to describe a new human protein, interferon beta-cis, isolated from primary cultures of human amniotic cells, the corresponding recombinant DNA molecule encoding it, and the process of production of the human recombinant protein interferon beta-cis produced through techniques of genetic engineering, to be used in human or veterinarian clinical applications or in research.